This invention relates to the quantitative determination of gamma glutamyl transpeptidase enzyme in biological fluids, particularly human blood serum, and to compositions employed in such assay.
The gamma glutamyl transpeptidase enzyme is present in various biological tissues, such as human and animal kidneys, as well as in urine and blood serum. Elevated levels of gamma glutamyl transpeptidase activity in serum is an indication of liver diseases, and extremely high levels have been associated with cancer of the liver, bile duct obstructions, and heart disorders of postmyocardial infarction. Thus, clinical determination of gamma glutamyl transpeptidase activity in serum has become a routine test in a large volume of pathological diagnosis. In the most widely employed method for determination of gamma glutamyl transpeptidase activity, the enzymatic conversion of the substrate gamma glutamyl-p-nitroanilide (GGpN) is employed. The liberated reaction product, p-nitroaniline, is a yellow-colored compound whose rate of formation is determined optically as the measure of the gamma glutamyl transpeptidase (GGTP) activity. The well known reaction ##STR1## was adapted to a clinical procedure for measurement of GGTP activity in serum by Szasz, Clinical Chemistry, 15,124 (1969) and optimized by the Scandinavian Society for Clinical Chemistry and Clinical Physiology, Scand. J. Clin. Lab. Invest., 36, 119 (1976). This method also has been adopted by the American Association for Clinical Chemistry.
One of the disadvantages of this standard procedure is the instability and poor aqueous solubility of the gamma glutamyl-p-nitroanilide substrate. Preparation of a reagent for the procedure encounters difficulty in dissolving the substrate which must be mixed with warm water and heated for five minutes at 50.degree.-60.degree. C. in a water bath or incubator. The reagent must then be cooled at room temperature to 37.degree. C. and used within one hour of cooling at 37.degree. C. in order to prevent precipitation. If precipitation occurs, the reagent must be reheated to 50.degree. C. and again cooled to 37.degree. C. for use. Consequently, it has been necessary to prepare solutions only large enough to perform relatively few determinations as soon as possible. Even with heating, the prepared substrate concentration can only be approximately 3 to 4 mmole/liter.
In the effort to avoid the poor solubility of the GGpN substrate, Rosalki and Tarlow dissolved the substrate in a dilute solution of HCl with limited stability (Clin. Chem., 20, 9 pp. 1121-1124 (1974). To eliminate the inconvenience required in using the HCl reagent, acidic and sulfonic derivatives of the substrate have been developed as described in U.S. Pat. Nos. 3,986,931 and 4,087,331, respectively. While these derivative substrates have improved aqueous solubility, their expense, slower rate of decomposition and multiplied reagent requirements have limited their clinical use. Consequently, GGpN has remained the substrate of choice for GGTP assay.
U.S. Pat. No. 3,878,048 describes the use of surfactants to solublize the GGpN substrate in basic media and once the substrate has been dissolved, the pH is adjusted to approximately 8.2 with addition of hydrochloric acid solution to promote the enzymatic reaction. Subsequent dye-coupling of the -p-nitroaniline reaction product is initiated under acidic condition, and the diazotized product is then optically measured for indication of the GGTP activity of the sample.
Prior art teaches use of a single, dry reagent which comprises GGpN substrate in a tablet or powder and the conventional glycylglycine acceptor for the liberated glutamyl, tris(hydroxymethyl)aminomethane and succinic acid to adjust the pH of the Tris-buffered reconstituted reagent to approximately 8.2. However, reconstitution of the single, dry reagent into the basic pH solution still requires dissolving in warm water at 37.degree. C. in order to form a stable solution. The resulting reagent solution is stable at room temperature for approximately 8 hours but cannot be refrigerated due to precipitation of the substrate.